The field of this invention is the area of plant molecular biology, and it relates in particular, to metal and organometal resistance genes functional in plants, transgenic plants containing same, and methods for remediation of environmental metal and organometal contamination using the transgenic plants of the present invention, especially using plants which detoxify organomercurial compounds.
Contamination of the environment with metal ions and/or organic, hydroxide and thiol derivatives of metals has increased over the last several decades, with toxic levels of the contaminants being reached in air, water and/or soil in certain locations. Contamination may stem from human and industrial sources, or in certain locales, the soil is contaminated naturally with such toxic metals as arsenic, cadmium, copper, cobalt, lead, mercury, selenium and/or zinc.
Mercury is often found in soil, aquatic and marine sediments as thiol salts, as chelates with acidic humic substances, as methylmercury and to a lesser extent other organomercurials, as hydroxides and as free Hg.sup.++. Mercury cycles through the aqueous phase and into the atmosphere as volatile elemental Hg and methylmercury, and is then oxidized and washed by rain into the marine environment [Barkay et al. (1992) Biodegradation 3:147-159]. Some bacteria in soil and sediments can detoxify ionic mercury by reducing it to its metallic form in an NADPH-coupled reaction, which is efficiently catalyzed by mercuric ion reductase.
Mercury is also often found bound in the form of organomercurial compounds in contaminated animals and microbes [Barkay et al. (1992) supra; Robinson and Tuovinen (1984) Microbiological Reviews 48:95-124]. Sulfate-reducing bacteria living in the aerobic-anaerobic interface in contaminated environments can convert mercuric ion to methyl mercury. In fish, where mercury toxicity is well studied, most of the tissue-associated mercury is found as methylmercury, and its production may be the product of nonenzymatic and enzymatic reaction of Hg.sup.++ with methyl-B12 [Pan Hou and Imura (1987) Arch. Microbiol. 131:176-177]. The prevailing theory, supported by convincing evidence, is that most methyl mercury is produced catalytically by sulfate-reducing bacteria living at the aerobic/anaerobic interface (Choi et al. (1994) Appl. Environ. Micro. 60, 1342-1346; Choi et al., (1994) Appl. Env. Microbiol. 60, 4072-4077). This activity is particularly high in aquatic environments, and hence the well-publicized link to the aquatic food chain (see below). Although both the mono- (CH.sub.3 Hg.sup.+) and dimethyl (Hg(CH.sub.3).sub.2) forms of methyl mercury are extremely toxic (Clarkson, T. W. (1994) In: Mercury Pollution Integration and Synthesis, C. J. Watras, and J. W. Huckabee, eds., Lewis Publishers Ann Arbor, Mich., pp. 631-642), the latter is very mobile in both liquid and gaseous phases and is more easily transported through cell membranes than thiol bound Hg(II) or free mercury ion. This mobility is apparently what makes methyl mercury such an environmental hazard. High levels of both thiol bound Hg(II) and methy mercury are found in organisms--worms, shrimp, crabs, bottom-feeding fish--that come in direct contact with contaminated sediment, but relatively little Hg(II) is found further up the food chain. However, methyl mercury is the principal mercury compound that is concentrated or "biomagnified" up the food chain from bacteria living on detritus at the sediment-water interface to bottom feeders and then on to fish, birds, and mammals (Gardner et al. (1978) Environ. Pollut. 15, 243-251). Methyl mercury has had a tragic impact on humans and animals, and it is often lethal. The first to show symptoms in the infamous Minamata Bay incident (D'Itri and D'Itri (1978) Environ. Management 2, 3-16) were birds and cats and then humans. The mercury found in all three species was be traced directly to methyl mercury-contaminated fish in the bay. The characterized neurological diseases and proposed immunological diseases produced by methyl mercury in animals and humans are so far untreatable. Methylmercury is volatile, and both mono- and dimethylmercury are extremely toxic [D'Itri and D'Itri (1987) Environ. Management 2:3-16].
The bacterial gene merA used by the present inventors is derived from the transposon Tn21, which was originally isolated from the Incompatibility Group IncFII resistance plasmid NR1 [see e.g., Gilbert and Summers (1988) Plasmid 20:127-136]. The product of the bacterial merA gene is mercuric ion reductase (MerA) . MerA can detoxify ionic mercury by reducing it to its less toxic (insoluble and volatile) elemental form (Hg.sup.0). MerA belongs to a family of reductase enzymes which are related in their primary structures. As a family, these reductases act on a wide variety of organic and thiol substrates in addition to the thiol salts of divalent Hg.
Certain bacterial mer operons also encode an organomercurial lyase (MerB, methylmercury lyase, R831b merB gene product) which catalyzes the protonolytic cleavage of carbon-mercury bonds, e.g., RCH.sub.2 Hg.sup.+ .fwdarw.Hg.sup.++ +RCH.sub.3, and together with MerA, produces what is termed broad spectrum mercury resistance (resistance to both thiolmercury and organomercurials including alkylmercury compounds and resistance to mercuric ion). The MerB protein cleaves a variety of carbon-mercury compounds, from methylmercury to long chain hydrocarbon and aromatic derivatives [Begley et al. (1986a) Biochemistry 25:7186-7192; Begley et al. (1986b) ibid. 7192-7200]. This process removes methylmercury or other organomercurials and then metallic mercury from the environment.
Additional genes which often occur within bacterial mer operons include merT (mercury transport through the cell membrane) and merP (mercury sequestration in the periplasmic space of gram-negative bacteria). Mercury resistance genes are reviewed in Summers, A. O. (1986) Ann. Rev. Microbiology 40:607-634.
Regions which are naturally contaminated with heavy metals are often characterized by scrubby heavy-metal tolerant vegetation [Brooks and Malaisse (1985) The Heavy Metal-tolerant Flora of South Central Africa, A.A. Balkema Press, Boston, Mass.; Wild, H. (1978) "The Vegetation of Heavy Metal and Other Toxic Soils," in Biogeography and Ecology of Southern Africa, Wergren, M. J. H., ed, Junk, The Hague, Netherlands]. Certain of these naturally occurring metal-resistant plants hyperaccumulate large amounts of heavy metals. These plants have been found in a variety of habitats, but often they exhibit bizarre metal ion requirements, grow poorly in less exotic habitats, and are of little direct economic value as crop or forest species.
There is a long felt need in the art for the in situ remediation of toxic metal ions and especially toxic organometal compounds (e.g., alkyl and/or aryl metal adducts). The present invention enables phytoremediation and/or revegetation of contaminated environments via the plant-expressible organometal lyase coding sequences, especially organomercurial lyases and mercuric ion reductase, alone or in combination with plant-expressible metal ion reductases, as disclosed herein.